Sunteți pe pagina 1din 20

NIH Public Access

Author Manuscript
JAMA. Author manuscript; available in PMC 2014 January 31.
Published in final edited form as:
NIH-PA Author Manuscript

JAMA. 2013 February 13; 309(6): 570577. doi:10.1001/jama.2012.155925.

ASSOCIATION BETWEEN MATERNAL USE OF FOLIC ACID


SUPPLEMENTS AND RISK OF AUTISM IN CHILDREN
Pl Surn, MD, MPHa,b, Christine Roth, MSca,c, Michaeline Bresnahan, PhDc,d, Margaretha
Haugen, PhDa, Mady Hornig, MDc, Deborah Hirtz, MDe, Kari Kveim Lie, MDa, W. Ian Lipkin,
MDc, Per Magnus, MD, PhDa, Ted Reichborn-Kjennerud, MD, PhDa,f, Synnve Schjlberg,
MSca, George Davey Smith, MD, DScg, Anne-Siri yen, PhDa,h, Ezra Susser, MD,
DrPH(*),c,d, and Camilla Stoltenberg, MD, PhDa,i,(*)
aThe Norwegian Institute of Public Health, Oslo, Norway

bCentre for Paediatric Epidemiology and Biostatistics, UCL Institute of Child Health, London,
United Kingdom
cThe Mailman School of Public Health, Columbia University, New York, NY, USA
NIH-PA Author Manuscript

dNew York State Psychiatric Institute, New York, NY, USA


eNational Institute of Neurological Disorders and Stroke, Bethesda, MD, USA
fInstitute of Psychiatry, University of Oslo, Oslo, Norway
gMRC Centre for Causal Analysis in Translational Epidemiology, University of Bristol, Bristol,
United Kingdom
hNic Waals Institute, Lovisenberg Hospital, Oslo, Norway
iDepartment of Public Health and Primary Health Care, University of Bergen, Bergen, Norway

Abstract
ContextPrenatal folic acid supplements reduce the risk of neural tube defects in children, but it
has not been determined whether they protect against other neurodevelopmental disorders.
ObjectiveTo examine the association between maternal use of prenatal folic acid supplements
and the subsequent risk of autistic disorder in children.
NIH-PA Author Manuscript

Design, Setting, and PatientsThe study sample of 85,176 was derived from the population-
based, prospective Norwegian Mother and Child Cohort Study (MoBa). The children were born in
200208. By the end of follow-up on March 31st, 2012, the age range was 3.310.2 years and the
mean age 6.4 years. The exposure of primary interest was use of folic acid from 4 weeks before to
8 weeks after the start of pregnancy. The start of pregnancy was defined as the first day of the last
menstrual period before conception. Relative risks of ASD were estimated by odds ratios (ORs)
with 95% confidence intervals (CIs) in a logistic regression analysis. Analyses were adjusted for
maternal education level, year of birth, and parity.
Main Outcome MeasureSpecialist-confirmed diagnosis of autistic disorder.
ResultsTo date, 114 children in the study sample have been diagnosed with autistic disorder.
In children whose mothers took folic acid, 0.10% (64/61,042) had autistic disorder, compared with
0.21% (50/24,134) in those unexposed to folic acid. The adjusted OR for autistic disorder in

Corresponding author: Pl Surn, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, NORWAY,
Telephone: (+47) 928 98 631, Fax: (+47) 22 35 36 05, pal.suren@fhi.no.
(*)Joint senior authorship
Surn et al. Page 2

children of folic acid users was 0.61 (95% CI, 0.410.90). Similar analyses for prenatal fish oil
supplements showed no such association with autistic disorder, even though fish oil use was
associated with the same maternal characteristics as folic acid use.
NIH-PA Author Manuscript

ConclusionPrenatal folic acid supplements around the time of conception were associated
with a lower risk of autistic disorder in the MoBa cohort.

INTRODUCTION
Supplementation with folic acid around the time of conception reduces the risk of neural
tube defects in children17. This protective effect has led to mandatory fortification of flour
with folic acid in several countries8, and it is generally recommended that women planning
to become pregnant take a daily supplement of folic acid from one month before
conception8,9.

There is also evidence that maternal folic acid supplementation during pregnancy may be
associated with reduced risk of other neurodevelopmental disorders in children. A recent
study of 38,954 children in the Norwegian Mother and Child Cohort Study (MoBa) found
that maternal intake of folic acid supplements from 4 weeks before to 8 weeks after the start
of pregnancy was associated with a lower risk of severe language delay at 3 years of age10.
A case-control study of autism spectrum disorder (ASD) from California showed that
maternal intake of folic acid and prenatal vitamins during the 3 months prior to pregnancy
NIH-PA Author Manuscript

and the first month of pregnancy was associated with a lower risk of ASD in the offspring,
and complementary genetic analyses indicated that the association was modified by gene
variants that determine the ability to utilize available folate11,12.

Although ethical considerations preclude placebo-controlled randomized trials that eliminate


folic acid, observational studies of mothers who do and do not use supplements may be
informative. Here we report the results of such an analysis in MoBa, wherein we
investigated the association between the use of maternal folic acid supplements ahead of and
in early pregnancy and the subsequent risk of autistic disorder in the offspring.

METHODS
Study Population
The MoBa13 cohort is nationwide and includes 109,000 children born from 1999 to 2009.
Mothers were recruited at ultrasound examinations around week 18 of gestation. Cases of
ASD in the cohort are identified by a sub-study of autism, the Autism Birth Cohort Study14.
The analyses in this study reflect data collected and processed by March 31st, 2012.
Participation in MoBa and the Autism Birth Cohort study is based on written informed
NIH-PA Author Manuscript

consent. Both studies are approved by the Regional Committee of Medical Research Ethics
for South-Eastern Norway.

Measures of ASD
Cases of ASD are identified through: 1) questionnaire screening of MoBa participants at the
ages of 36 months, 5 years and 7 years, 2) professional and parental referrals of participants
suspected of having ASD, and 3) linkages to the Norwegian Patient Registry. Referrals are
elicited through annual newsletters to MoBa participants and information on the Norwegian
Institute of Public Health website. The Norwegian Patient Registry collects data on
diagnoses from all hospitals and outpatient clinics in Norway, beginning in the year 2008,
thereby capturing all children diagnosed with ASD by Norwegian health services.

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 3

When a child with ASD or potential ASD is detected through any of the mechanisms
described above, he/she is invited to participate in a clinical assessment that includes the
research-standard instruments for diagnosis of ASD, the Autism Diagnostic Interview
NIH-PA Author Manuscript

Revised (ADI-R)15 and the Autism Diagnostic Observation Schedule (ADOS)16, which
have proven high reliability and validity in making diagnoses of ASD in children.
Assessments are conducted without knowledge of previous questionnaire responses.
Diagnostic conclusions are best-estimate clinical diagnoses derived from test and interview
results and from information collected from parents and teachers. Diagnoses are based on
DSM-IV criteria, and the case definition includes 299.00 Autistic Disorder, 299.80
Aspergers Syndrome, and 299.80 Pervasive Developmental Disorder Not Otherwise
Specified (PDD-NOS).

The registry contains ICD-10 codes determined by Norwegian specialist health services, and
the ASD case definition of the Autism Birth Cohort study includes F84.0 Childhood Autism,
F84.1 Atypical Autism, F84.5 Aspergers Syndrome, F84.8 Other Pervasive Developmental
Disorder, and F84.9 Pervasive Developmental Disorder, Unspecified. In this paper, we have
used the term autistic disorder for F84.0 and PDD-NOS for F84.1, F84.8, and F84.9.

Measures of Folic Acid Use and Dietary Folate Intake


Since 1998, the Norwegian Directorate of Health has recommended that all women
attempting to become pregnant should take a supplement of 400 g folic acid per day from
NIH-PA Author Manuscript

one month before conception through the first trimester. Folic acid supplements are
available over the counter in Norway. There are also multivitamin supplements with folic
acid available, but at the time of recruitment to MoBa, all such supplements contained less
than 400 g of folic acid.

In MoBa, detailed information about the mothers supplement intake before conception and
in early pregnancy was obtained through questionnaire report at week 18 of gestation. No
foods were fortified with folic acid at the time when participants were recruited; synthetic
supplements thus represented the only source of folate apart from the ordinary diet for the
pregnant women. The women were asked to record their intake of vitamins, minerals and
other supplements according to the ingredient lists on the supplement containers, within 4-
week intervals from before the start of pregnancy. They were not asked to specify the exact
amounts, so if folic acid was only taken as part of a multivitamin supplement, the daily dose
would be lower than 400 g.

Additional information about supplement use and dietary intake in mid-pregnancy was
obtained through a food-frequency questionnaire completed in week 22. In this
questionnaire, women were asked to write the name of supplements that they were currently
NIH-PA Author Manuscript

taking (in week 22), and exact amounts of vitamins and minerals were calculated on the
basis of this information. The food-frequency questionnaire has been described in a previous
paper17 and validated through blood samples and 4-day food records from a subsample of
the cohort18.

Measures of Timing
For our primary analyses, we examined an interval from 4 weeks before to 8 weeks after the
start of pregnancy. The start of pregnancy was defined as the first day of the last menstrual
period before conception, in keeping with the standard definition used in the follow-up of
pregnant women in Norway. Children of mothers who used folic acid supplements during
the entire or parts of the exposure interval were compared to children whose mothers did not
use folic acid supplements during the interval. The exposure interval was chosen on the
basis of an a priori hypothesis that the effect of folic acid on the development of the central

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 4

nervous system is most prominent in this period, and it also corresponds to the interval used
in the previous study of language delay10. The interval covers or precedes events of critical
importance to the fetal brain, such as the closure of the neural tube 28 days after conception
NIH-PA Author Manuscript

(gestational week 6) and the embryonic period with the development of the basic brain
structures 1556 days after conception (gestational week 510)19.

Potential Confounders
We explored a number of factors that might influence a potential association between
supplement use and ASD risk: parental education, parental age, whether the pregnancy was
planned, maternal smoking during pregnancy, maternal body mass index (weight in
kilograms divided by height in meters squared), parity, and year of birth.

Statistical Analyses
Analyses were done using SPSS version 19.0 (SPSS Inc., Chicago, Illinois). Odds ratios
(ORs), with 95% confidence intervals (CIs), were estimated from logistic regression models.
The adjusted models included adjustment for year of birth, maternal education level, and
parity, as these were the only covariates that had any influence on the OR estimates. We
estimated the power of these analyses for autistic disorder, which was the ASD subtype with
the highest number of diagnosed cases in the study sample. The power calculations were
based on the observed distributions of the outcome and the exposure, i.e., an overall
NIH-PA Author Manuscript

prevalence of 0.13% of autistic disorder and a proportion of 68% of the study sample
exposed to folic acid within the exposure interval. The type I error probability was set at
=0.05 (2-sided). Under these conditions we had a power of 93% to detect an OR of 0.50,
73% for OR 0.60, 45% for OR 0.70, and 18% for OR 0.80.

We conducted a secondary analysis of the association between maternal use of fish oil
supplements and the risk of ASD, in order to investigate whether the associations were
specific to folic acid or similar across different types of supplements. If they were similar,
the associations would more likely be attributable to health-conscious maternal behaviors in
general and not the supplements per se20. We also examined the association between folic
acid use in week 22 of pregnancy and subsequent risk of ASD, to evaluate whether any
associations, if present, were similar in early pregnancy and mid-pregnancy.

The study did not have sufficient power for subgroup analyses, but we have included results
of some exploratory analyses for the autistic disorder subtype, as they provide valuable
additional information and clues to interactions that could be tested in future studies. We
explored the following:
the timing of initiation of folic acid supplementation
NIH-PA Author Manuscript

maternal use of other vitamins/minerals from 4 weeks before to 8 weeks after the
start of pregnancy
maternal total daily intake of folate in week 22 (diet and supplements combined,
adjusted for dietary folate equivalents)
stratification of autistic disorder cases by language level at 36 months
stratification of the study sample by year of birth (200204 vs. 200508)

RESULTS
The derivation of the study sample is described in Figure 1. A total of 97,179 cohort
participants were eligible for the analyses. To isolate folic acid exposure from other
exposure reported to increase the risk of ASD, we excluded children with gestational age

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 5

<32 weeks at birth, children with birth weight <2,500 g, and multiple births. We also
excluded children for whom we did not have data on maternal supplement use before
conception and in early pregnancy, and children whose mothers reported supplement use but
NIH-PA Author Manuscript

had not specified the type and duration. In total, 12,003 children were excluded, for one or
more reasons. The final study sample included 85,176 children. At the end of follow-up, the
age range was 3.310.2 years and the mean age 6.4 years.

A total of 270 children (0.32%) in the study sample have been diagnosed with ASD: 114
(0.13%) with autistic disorder, 56 (0.07%) with Aspergers syndrome, and 100 (0.12%) with
PDD-NOS. The distribution of ASD cases by year of birth is shown in eTable 1 of the
online supplement. Of the ASD cases, 135 (50.0%) had been clinically assessed through the
ABC study. The remaining 135 had specialist-confirmed diagnoses of ASD recorded in the
Norwegian Patient Registry. Registry diagnoses have a high validity for ASD as a whole: of
the 39 children assessed in the ABC study after being detected through the registry, 38 were
found to meet DSM-IV criteria for ASD, generating a positive predictive value (PPV) of
97% (95% CI, 87100%). PPV estimates are lower for the individual ASD subtype
diagnoses: 80% (12/15) for autistic disorder (95% CI, 5296%), 38% (5/13) for Aspergers
syndrome (95% CI, 1468%), and 73% (8/11) for PDD-NOS (95% CI, 3994%). PPV
estimates for the subtype diagnoses are preliminary, as the number of cases in each group is
still low.
NIH-PA Author Manuscript

The proportions of mothers reporting folic acid use are shown in Figure 2. In the first
interval (weeks 4 to 1 before the start of pregnancy), 32.9% of mothers took folic acid. The
proportion increased to 70.7% in week 912 and then reverted to 45.8% in week 1317. The
distribution of folic acid use across categories of parent and child characteristics is shown in
Table 1. Women who used folic acid within the exposure interval (4 weeks before to 8
weeks after the start of pregnancy) were more likely to have college or university level
education, to have planned the pregnancy, to be non-smokers, to have pre-pregnancy BMI
below 25, and to be first-time mothers. Folic acid use increased substantially by year of
birth, from 43.2% in 2002 to 83.7% in 2008.

Women who took folic acid in early pregnancy had a higher response rate to the screening
questionnaire completed when the children were 36 months. For the study sample overall,
the response rate was 62% in folic acid users and 55% in non-users. For children born in
200508, i.e., the youngest children, the difference was somewhat larger, with a response
rate of 61% in folic acid users and 50% in non-users. Consequently, ASD children born to
women who used folic acid may have had a higher probability of being diagnosed at an
early age.
NIH-PA Author Manuscript

Results of the logistic regression analysis for autistic disorder are displayed in Table 2.
There was an inverse association between folic acid use and the subsequent risk of autistic
disorder. In children whose mothers took folic acid, 0.10% (64/61,042) had autistic disorder,
compared with 0.21% (50/24,134) in children whose mothers did not take folic acid. The
adjusted OR of autistic disorder was 0.61 (95% CI, 0.410.90) in children of folic acid
users.

The use of fish oil supplements followed similar patterns as folic acid use in the study
sample: it was associated with the same parental characteristics (eTable 2), it increased
throughout the period of recruitment to the cohort (eTable 2), and it increased from before
pregnancy through the first trimester (eFigure 1). Despite these similarities, there was no
association between fish oil supplement use and autistic disorder risk, as shown in Table 3.
The adjusted OR of autistic disorder was 1.29 (95% CI, 0.881.89) in children of mothers
who used fish oil supplements.

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 6

The inverse association found for folic acid use in early pregnancy was absent for folic acid
use in mid-pregnancy: the adjusted OR for autistic disorder was 0.96 (95% CI, 0.601.55)
for those taking 400 g per day in week 22, and 1.02 (95% CI, 0.621.67) for those taking
NIH-PA Author Manuscript

less than 400 g per day at that time.

For Aspergers syndrome and PDD-NOS, we restricted the analyses to birth years with a
cumulative incidence of 0.08% or higher (higher than the lowest level observed for autistic
disorder): 200204 for Aspergers syndrome (n=30,117 including 48 cases) and 200206 for
PDD-NOS (n=59,152 including 91 cases). Our power to detect an OR of similar magnitude
to that found for autistic disorder (OR=0.61) was limited: 36% for Aspergers syndrome,
and 61% for PDD-NOS. For Aspergers syndrome, the proportion of diagnosed cases was
0.12% (21/17,218) in children of folic acid users and 0.21% (27/12,899) in children of non-
users, generating an adjusted OR of 0.65 (95% CI, 0.361.16). For PDD-NOS, the
proportion was 0.15% (58/39,543) in children of folic acid users and 0.17% (33/19,649) in
children of non-users, generating an adjusted OR of 1.04 (95% CI, 0.661.63).

Results of exploratory analyses are displayed in Table 4. They should be cautiously


interpreted, as none of the exploratory analyses had a statistical power of more than 50% to
detect an OR of 0.60. There did not seem to be a strong gradient in risk by timing of
initiation of folic acid use within the primary exposure interval (Table 4, analysis #1). The
use of other vitamins and minerals in addition to folic acid did not appear to affect autistic
NIH-PA Author Manuscript

disorder risk (Table 4, analysis #2). The analyses based on the food-frequency questionnaire
data from week 22 did not reveal any apparent association between maternal total daily
folate intake in week 22 (diet and supplements combined) and subsequent risk of autistic
disorder in children (Table 4, analysis #3). The analysis in which cases were stratified
according to language level suggested that the inverse association may be strong in those
with severe language delay and weak in those with moderate or no delay (Table 4, analysis
#4). The analysis stratified by year of birth suggested that the inverse association may be
stronger in the older children (born in 200204) than in the younger children (born in 2005
08) (Table 4, analysis #5).

COMMENT
The study found that maternal folic acid supplementation from 4 weeks before to 8 weeks
after the start of pregnancy was associated with a lower risk of autistic disorder the most
severe form of ASD in children. If the observed inverse association represents a causal
relationship, the finding indicates that a deficiency of folate around conception and early
pregnancy, or a reduced ability to utilize available folate, are important causes of autistic
disorder. If so, this finding creates opportunities for highly effective preventive measures.
NIH-PA Author Manuscript

Use of folic acid supplements was associated with higher socio-economic status and more
health-conscious maternal behavior patterns in the study sample. We cannot exclude the
possibility that some portion of the inverse association represents residual, unmeasured
confounding. However, if residual confounding was substantial, we would have expected to
find a lowering of risk associated with fish oil supplement use as well, since the use of fish
oil was associated with exactly the same parental characteristics in the study sample. No
such lowering of risk was observed. We would also have expected the inverse association
between folic acid use and autistic disorder risk to persist in mid-pregnancy (week 22),
which it did not.

To further assess the possibility of residual confounding, we explored whether maternal


illness and medication use during pregnancy had any effect on the inverse association.
Information about maternal illness and medication use was obtained from the questionnaire
completed in week 18 and from the Medical Birth Registry. We adjusted the logistic

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 7

regression models for the presence of anxiety, depression, epilepsy, preeclampsia, and
diabetes during pregnancy (separately for each disorder). We also made separate
adjustments for use of medications for anxiety, depression, and epilepsy, and for the use of
NIH-PA Author Manuscript

hormone treatment and in vitro fertilization to become pregnant. None of the adjustments
made any difference, which might reflect the fact that the pregnant women in the cohort
were generally healthy and had low proportions of medication use during pregnancy. We did
not have data on more rare psychiatric disorders, but we believe that such disorders are
unlikely to have had any significant influence.

Our findings indicate that the inverse association may be largely driven by the autistic
disorder cases with severe language delay at 36 months, who were presumably the more
severely affected children. It is also worth noting that the OR estimate for those with
missing data on language level (non-responders to the screening questionnaire) was similar
to those with severe language delay. This suggests that mothers with severely affected
children may have had lower response rates, and that an ascertainment bias may have been
present. The possibility of such bias, combined with the small numbers in each stratum,
warrants caution in the interpretation of these findings.

The participation rate among women invited to participate in MoBa was 38.5%, and the
cohort is not fully representative of the Norwegian population. Comparisons to nationwide
registry data have demonstrated that the mothers in the cohort were more likely to be first-
NIH-PA Author Manuscript

time mothers and that they had higher levels of education, a higher mean age, and lower
levels of smoking than other pregnant women21. The proportions of single mothers and
mothers with an immigrant background were very low (unpublished data). We tested the
generalizability of our findings by replicating the analyses in a nationwide data file
containing data from the Medical Birth Registry of Norway, the Norwegian Patient Registry,
and Statistics Norway. We included children born in 19992007 and applied the same
inclusion criteria as for the MoBa-based analyses. The replication sample included 473,095
children, of whom 822 (0.17%) had autistic disorder diagnoses recorded by Norwegian
specialist health services. Folic acid use is substantially underreported in the Medical Birth
Registry; our comparisons with the MoBa questionnaire data found that half of the mothers
reported to be non-users in the registry actually had reported folic acid use in the
questionnaires. This underreporting is a major limitation and will bias association measures
towards the null. Despite this, we did find a significant inverse association in the nationwide
sample: mothers with reported folic acid use had an adjusted OR of 0.83 (95% CI, 0.71
0.97) for autistic disorder in their children. When the MoBa participants were analysed using
the registry-based folic acid variable, the adjusted OR was 0.75 (95% CI, 0.460.96). The
similarity between MoBa participants and non-participants indicate that our MoBa-based
analyses have not been significantly affected by selection bias.
NIH-PA Author Manuscript

The strengths of the study were the cohort design, large sample size, population-based
recruitment of cohort participants, prospective data collection, and the combination of
screening, referrals and registry linkage for detection of ASD cases. The richness of the
exposure data allowed for differentiations between different supplements, and between the
various stages of pregnancy. Our ability to compare the study sample with a nationwide
sample was also an advantage. The main limitation was the incomplete ascertainment of
ASD cases in the cohort. The prevalence of diagnosed ASD was lower than what has been
reported from the United Kingdom and the United States22,23, although that discrepancy is
not merely due to underscertainment, because the nationwide ASD prevalence is also lower
in Norway24. For the country as a whole, the prevalence is estimated to be 0.8% in 12-year-
olds24, which is not very different from the 0.66% prevalence observed for children born in
2002 and 2003 in the MoBa cohort (eTable 1). Underascertainment was less of a problem
for autistic disorder than for the other ASD subtypes, but it was still reassuring that the

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 8

inverse association for autistic disorder was stronger in the older children (born in 200204),
for whom case ascertainment was closer to completion. The relative weakness of the inverse
association among the younger children (born in 200508) may have resulted from the fact
NIH-PA Author Manuscript

that mothers who took prenatal folic acid supplements had higher questionnaire response
rates, causing the ASD cases among their children to be identified earlier. As mentioned
previously, there was also the possibility of ascertainment bias arising from lower response
rates among parents of the more severely affected autistic disorder cases. If such bias was
present, the OR estimate for the younger children would be biased towards the null..

Another limitation of the study was the reliance on subtype diagnoses of ASD. These have
not been found to have high reliability across assessment sites in studies in the United
States, and may be removed altogether from the upcoming DSM-V classification system25.
Our own validation of registry diagnoses indicated that subtype diagnoses were less reliable
than for ASD as a whole, but there was still a high level of agreement (PPV=80%) for
autistic disorder diagnoses, which was the outcome of primary interest.

Our main finding was that maternal use of folic acid supplements around the time of
conception was associated with a lower risk of autistic disorder. This finding does not
establish a causal relation between folic acid use and autistic disorder, but provides a
rationale for replicating the analyses in other study samples and further investigating genetic
factors and other biological mechanisms that may explain the inverse association.
NIH-PA Author Manuscript

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
The Norwegian Mother and Child Cohort is supported by the Norwegian Ministry of Health and Care Services, the
Norwegian Ministry of Education and Research, the Research Council of Norway/FUGE (grant 151918), the
National Institute of Neurological Disorders and Stroke (NIH/NINDS), Bethesda, MD, USA (grant NS47537
[Lipkin]), and the National Institute of Environmental Health Sciences (NIH/NIEHS), Research Triangle Park, NC,
USA (contract NO-ES-75558). The Autism Birth Cohort study is funded by the NINDS (grant NS47537 [Lipkin]).
Pl Surns salary is funded by the Research Council of Norway (grants 185476 and 190694). We are grateful to all
the families in Norway who take part in these ongoing studies. Camilla Stoltenberg and Ezra Susser declare that
they had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy
of the data analysis. Deborah Hirtz, the Scientific Program Officer for the Autism Birth Cohort study at NINDS,
participated in the design and conduct of the study; collection, management, analysis, and interpretation of the data;
and preparation, review, and approval of the manuscript.

Abbreviations
NIH-PA Author Manuscript

ASD Autism spectrum disorder


CI Confidence interval
MoBa The Norwegian Mother and Child Cohort Study
OR Odds ratio
PDD-NOS Pervasive developmental disorder not otherwise specified
PPV Positive predictive value

References
1. Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional
vitamin supplementation. N Engl J Med. Dec 24; 1992 327(26):18321835. [PubMed: 1307234]

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 9

2. Werler MM, Shapiro S, Mitchell AA. Periconceptional folic acid exposure and risk of occurrent
neural tube defects. JAMA. Mar 10; 1993 269(10):12571261. [PubMed: 8437302]
3. Daly LE, Kirke PN, Molloy A, Weir DG, Scott JM. Folate levels and neural tube defects.
NIH-PA Author Manuscript

Implications for prevention. JAMA. Dec 6; 1995 274(21):16981702. [PubMed: 7474275]


4. Shaw GM, Schaffer D, Velie EM, Morland K, Harris JA. Periconceptional vitamin use, dietary
folate, and the occurrence of neural tube defects. Epidemiology. May; 1995 6(3):219226.
[PubMed: 7619926]
5. Berry RJ, Li Z, Erickson JD, et al. Prevention of neural-tube defects with folic acid in China. China-
U.S. Collaborative Project for Neural Tube Defect Prevention. N Engl J Med. Nov 11; 1999
341(20):14851490. [PubMed: 10559448]
6. Milunsky A, Jick H, Jick SS, et al. Multivitamin/folic acid supplementation in early pregnancy
reduces the prevalence of neural tube defects. JAMA. Nov 24; 1989 262(20):28472852. [PubMed:
2478730]
7. MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical
Research Council Vitamin Study. Lancet. Jul 20; 1991 338(8760):131137. [PubMed: 1677062]
8. Centers for Disease C, Prevention. CDC Grand Rounds: additional opportunities to prevent neural
tube defects with folic acid fortification. MMWR Morbidity and mortality weekly report. Aug 13;
2010 59(31):980984. [PubMed: 20703205]
9. Scientific Advisory Committee on Nutrition. London, United Kingdom: 2006. Folate and Disease
Prevention.
10. Roth C, Magnus P, Schjolberg S, et al. Folic acid supplements in pregnancy and severe language
NIH-PA Author Manuscript

delay in children. JAMA. Oct 12; 2011 306(14):15661573. [PubMed: 21990300]


11. Schmidt RJ, Hansen RL, Hartiala J, et al. Prenatal vitamins, one-carbon metabolism gene variants,
and risk for autism. Epidemiology. Jul; 2011 22(4):476485. [PubMed: 21610500]
12. Schmidt RJ, Tancredi DJ, Ozonoff S, et al. Maternal periconceptional folic acid intake and risk of
autism spectrum disorders and developmental delay in the CHARGE (CHildhood Autism Risks
from Genetics and Environment) case-control study. The American journal of clinical nutrition.
Jul; 2012 96(1):8089. [PubMed: 22648721]
13. Magnus P, Irgens LM, Haug K, Nystad W, Skjaerven R, Stoltenberg C. Cohort profile: the
Norwegian Mother and Child Cohort Study (MoBa). Int J Epidemiol. Oct; 2006 35(5):11461150.
[PubMed: 16926217]
14. Stoltenberg C, Schjolberg S, Bresnahan M, et al. The Autism Birth Cohort: a paradigm for gene-
environment-timing research. Mol Psychiatry. Jul; 2010 15(7):676680. [PubMed: 20571529]
15. Lord C, Rutter M, Le Couteur A. Autism Diagnostic Interview-Revised: a revised version of a
diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.
J Autism Dev Disord. Oct; 1994 24(5):659685. [PubMed: 7814313]
16. Lord C, Risi S, Lambrecht L, et al. The autism diagnostic observation schedule-generic: a standard
measure of social and communication deficits associated with the spectrum of autism. J Autism
Dev Disord. Jun; 2000 30(3):205223. [PubMed: 11055457]
NIH-PA Author Manuscript

17. Meltzer HM, Brantsaeter AL, Ydersbond TA, Alexander J, Haugen M. Methodological challenges
when monitoring the diet of pregnant women in a large study: experiences from the Norwegian
Mother and Child Cohort Study (MoBa). Matern Child Nutr. Jan; 2008 4(1):1427. [PubMed:
18171404]
18. Brantsaeter AL, Haugen M, Alexander J, Meltzer HM. Validity of a new food frequency
questionnaire for pregnant women in the Norwegian Mother and Child Cohort Study (MoBa).
Matern Child Nutr. Jan; 2008 4(1):2843. [PubMed: 18171405]
19. Sadler, TW. Langmans Medical Embryology. Baltimore, MD: Lippincott Williams & Wilkins;
2010.
20. Smith GD. Assessing intrauterine influences on offspring health outcomes: can epidemiological
studies yield robust findings? Basic & clinical pharmacology & toxicology. Feb; 2008 102(2):
245256. [PubMed: 18226080]
21. Nilsen RM, Vollset SE, Gjessing HK, et al. Self-selection and bias in a large prospective
pregnancy cohort in Norway. Paediatric and perinatal epidemiology. Nov; 2009 23(6):597608.
[PubMed: 19840297]

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 10

22. Baird G, Simonoff E, Pickles A, et al. Prevalence of disorders of the autism spectrum in a
population cohort of children in South Thames: the Special Needs and Autism Project (SNAP).
Lancet. Jul 15; 2006 368(9531):210215. [PubMed: 16844490]
NIH-PA Author Manuscript

23. Prevalence of autism spectrum disorders--Autism and Developmental Disabilities Monitoring


Network, 14 sites, United States, 2008. MMWR Surveill Summ. Mar 30; 2012 61(3):119.
24. Suren P, Bakken IJ, Aase H, et al. Autism spectrum disorder, ADHD, epilepsy, and cerebral palsy
in Norwegian children. Pediatrics. Jul; 2012 130(1):e152158. [PubMed: 22711729]
25. Lord C, Petkova E, Hus V, et al. A multisite study of the clinical diagnosis of different autism
spectrum disorders. Arch Gen Psychiatry. Mar; 2012 69(3):306313. [PubMed: 22065253]
NIH-PA Author Manuscript
NIH-PA Author Manuscript

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 11
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 12
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 1.
Derivation of the Study Sample
NIH-PA Author Manuscript

JAMA. Author manuscript; available in PMC 2014 January 31.


Surn et al. Page 13
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 2.
Folic Acid Supplement Use by Pregnancy Interval
NIH-PA Author Manuscript

JAMA. Author manuscript; available in PMC 2014 January 31.


NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Table 1

Parent and Child Characteristics by Maternal Folic Acid Use a

Supplement Use Week (4) to 8


Surn et al.

Total Study Sample (n=85,176) No Folic Acid (n=24,134) Use of Folic Acid (n=61,042)

n % n % n %

Maternal education, y
< 12 6,264 7.4 % 3,274 13.6 % 2,990 4.9 %
12 22,616 26.6 % 8,319 34.5 % 14,297 23.4 %
13 16 34,641 40.7 % 8,298 34.4 % 26,343 43.2 %
>= 17 19,910 23.4 % 3,611 15.0 % 16,299 26.7 %
Missing data 1,745 2.0 % 632 2.6 % 1,113 1.8 %

Paternal education, y
< 12 9,067 10.6 % 3,915 16.2 % 5,152 8.4 %
12 32,161 37.8 % 10,434 43.2 % 21,727 35.6 %
13 16 22,476 26.4 % 5,248 21.7 % 17,228 28.2 %
>= 17 18,835 22.1 % 3,541 14.7 % 15,294 25.1 %
Missing data 2,637 3.1 % 996 4.1 % 1,641 2.7 %

Maternal age, y
< 25 9,384 11.0 % 3,785 15.7 % 5,599 9.2 %
25 29 28,107 33.0 % 7,450 30.9 % 20,657 33.8 %

JAMA. Author manuscript; available in PMC 2014 January 31.


30 34 32,975 38.7 % 8,551 35.4 % 24,424 40.0 %
>= 35 14,710 17.3 % 4,348 18.0 % 10,362 17.0 %

Paternal age, y
< 25 4,041 4.7 % 1,709 7.1 % 2,332 3.8 %
25 29 19,167 22.5 % 5,331 22.1 % 13,836 22.7 %
30 34 33,101 38.9 % 8,629 35.8 % 24,472 40.1 %
35 39 20,231 23.8 % 5,644 23.4 % 14,587 23.9 %
>= 40 8,411 9.9 % 2,734 11.3 % 5,677 9.3 %
Missing data 225 0.3 % 87 0.4 % 138 0.2 %
Page 14
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Supplement Use Week (4) to 8

Total Study Sample (n=85,176) No Folic Acid (n=24,134) Use of Folic Acid (n=61,042)

n % n % n %
Surn et al.

Planned pregnancy
Yes 68,094 79.9 % 17,394 72.1 % 50,700 83.1 %
No 16,061 18.9 % 6,351 26.3 % 9,710 15.9 %
Missing data 1,021 1.2 % 389 1.6 % 632 1.0 %

Maternal smoking b
No 77,845 91.4 % 20,641 85.5 % 57,204 93.7 %
Yes 6,740 7.9 % 3,254 13.5 % 3,486 5.7 %
Missing data 591 0.7 % 239 1.0 % 352 0.6 %

Maternal pre-pregnancy BMI


< 25 56,614 66.5 % 15,150 62.8 % 41,464 67.9 %
25 29 18,332 21.5 % 5,441 22.5 % 12,891 21.1 %
30 34 5,830 6.8 % 1,951 8.1 % 3,879 6.4 %
>= 35 2,158 2.5 % 719 3.0 % 1,439 2.4 %
Missing data 2,242 2.6 % 873 3.6 % 1,369 2.2 %

Parity c
0 37,946 44.6 % 9,266 38.4 % 28,680 47.0 %

JAMA. Author manuscript; available in PMC 2014 January 31.


1 30,674 36.0 % 8,493 35.2 % 22,181 36.3 %
>= 2 16,556 19.4 % 6,375 26.4 % 10,181 16.7 %

Year of birth n Column % n Row % n Row %


2002 7,377 8.7 % 4,187 56.8 % 3,190 43.2 %
2003 10,858 12.7 % 5,030 46.3 % 5,828 53.7 %
2004 11,882 13.9 % 3,682 31.0 % 8,200 69.0 %
2005 13,711 16.1 % 3,504 25.6 % 10,207 74.4 %
2006 15,364 18.0 % 3,246 21.1 % 12,118 78.9 %
2007 14,140 16.6 % 2,552 18.0 % 11,588 82.0 %
2008 11,844 13.9 % 1,933 16.3 % 9,911 83.7 %
Page 15
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
a
p<0.001 for all variables, 2-sided chi-square test for independence.
b
Maternal smoking, daily or occasionally, during pregnancy.
c
Parity including previous miscarriage or abortion after week 22 of gestation.
Surn et al.

JAMA. Author manuscript; available in PMC 2014 January 31.


Page 16
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Table 2
Risk of Autistic Disorder According to Maternal Folic Acid Use

Total Autistic Disorder Unadjusted Adjusted a


Surn et al.

n % n % OR 95% CI OR 95% CI
No folic acid 24,134 28.3 % 50 0.21 % 1 (ref) --- 1 (ref) ---
Use of folic acid 61,042 71.7 % 64 0.10 % 0.51 0.35 0.73 0.61 0.41 0.90

a
Adjusted for year of birth, maternal education level, and parity. For maternal education, missing data was included as a separate category in the logistic regression model.

JAMA. Author manuscript; available in PMC 2014 January 31.


Page 17
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Table 3
Secondary Analyses

Total Autistic Disorder Unadjusted Adjusted a


Surn et al.

n % n % OR 95% CI OR 95% CI

Fish oil use in week (4) to 8 b


No fish oil 46,314 54.4 % 60 0.13 % 1 (ref) --- 1 (ref) ---
Use of fish oil 38,862 45.6 % 54 0.14 % 1.07 0.74 1.55 1.29 0.88 1.89

Folic acid use in week 22 c


No folic acid 32,064 40.4 % 42 0.13 % 1 (ref) --- 1 (ref) ---
1399 g/day 20,872 26.3 % 26 0.12 % 0.95 0.58 1.55 1.02 0.62 1.67
400 g/day 26,467 33.3 % 31 0.12 % 0.89 0.56 1.42 0.96 0.60 1.55

a
Adjusted for year of birth, maternal education level, and parity. For maternal education, missing data was included as a separate category in the logistic regression model.
b
Fish oil includes cod liver oil and omega-3 fatty acid supplements.
c
Analyses includes only responders to the food-frequency questionnaire in week 22 (n=79,403). The food-frequency questionnaire included report of brand names of supplements, enabling exact
calculations of the amounts of folic acid ingested per day.

JAMA. Author manuscript; available in PMC 2014 January 31.


Page 18
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Table 4
Exploratory Analyses

Total Autistic Disorder Unadjusted Adjusted a


Surn et al.

n % n % OR 95% CI OR 95% CI

1. Initiation of folic acid


No folic acid 14,721 17.3 % 32 0.22 % 1 (ref) --- 1 (ref) ---
Initiation week (4) to (1) 28,061 32.9 % 32 0.11 % 0.52 0.32 0.86 0.67 0.40 1.14
Initiation week 0 to 4 16,797 19.7 % 18 0.11 % 0.49 0.28 0.88 0.58 0.32 1.05
Initiation week 5 to 8 16,184 19.0 % 14 0.09 % 0.40 0.21 0.75 0.44 0.23 0.83
Initiation week 9 to 17 9,395 11.0 % 18 0.19 % 0.88 0.49 1.57 0.87 0.49 1.57

2. Use of other vitamins and minerals in week (4) to 8


No vitamins/minerals 21,106 24.8 % 44 0.21 % 1 (ref) --- 1 (ref) ---
Other vitamins/minerals, no folic acid 3,028 3.6 % 6 0.20 % 0.95 0.41 2.23 0.93 0.40 2.19
Folic acid only 29,075 34.1 % 31 0.11 % 0.51 0.32 0.81 0.62 0.38 1.00
Folic acid plus other vitamins/minerals 31,967 37.5 % 33 0.10 % 0.50 0.32 0.78 0.58 0.36 0.94

3. Total daily folate intake in week 22 (g) b


Quartile 1 (mean 209, range 62272) 19,611 24.7 % 26 0.13 % 1 (ref) --- 1 (ref) ---
Quartile 2 (mean 345, range 272434) 19,634 24.7 % 22 0.11 % 0.85 0.48 1.49 0.88 0.50 1.55
Quartile 3 (mean 560, range 434667) 19,485 24.5 % 26 0.13 % 1.01 0.58 1.73 1.08 0.62 1.87
Quartile 4 (mean 874, range 6675,673) 19,429 24.5 % 23 0.12 % 0.89 0.51 1.57 0.97 0.55 1.72

JAMA. Author manuscript; available in PMC 2014 January 31.


Intake not quantified c 1,244 1.6 % 2 0.16 % 1.21 0.29 5.12 1.01 0.24 4.27

4. Autistic disorder cases stratified by language level at 36 months d


Severe language delay
No folic acid 24,134 28.3 % 18 0.07 % 1 (ref) --- 1 (ref) ---
Use of folic acid 61,042 71.7 % 18 0.03 % 0.40 0.21 0.76 0.49 0.25 0.99
Moderate or no language delay
No folic acid 24,134 28.3 % 13 0.05 % 1 (ref) --- 1 (ref) ---
Use of folic acid 61,042 71.7 % 29 0.05 % 0.88 0.46 1.70 0.91 0.46 1.81
Missing data
Page 19
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Total Autistic Disorder Unadjusted Adjusted a

n % n % OR 95% CI OR 95% CI
No folic acid 24,134 28.3 % 19 0.08 % 1 (ref) --- 1 (ref) ---
Surn et al.

Use of folic acid 61,042 71.7 % 17 0.03 % 0.35 0.18 0.68 0.48 0.24 0.96

5. Study sample stratified by year of birth


200204
No folic acid 12,899 42.8 % 37 0.29 % 1 (ref) --- 1 (ref) ---
Use of folic acid 17,218 57.2 % 21 0.12 % 0.42 0.25 0.73 0.45 0.26 0.79
200508
No folic acid 11,235 20.4 % 13 0.12 % 1 (ref) --- 1 (ref) ---
Use of folic acid 43,824 79.6 % 43 0.10 % 0.85 0.46 1.58 0.84 0.45 1.59

a
Adjusted for year of birth, maternal education level, and parity. For maternal education, missing data was included as a separate category in the logistic regression models. The analysis stratified by year of
birth was adjusted for maternal education level and parity.
b
Analyses includes only responders to the food-frequency questionnaire in week 22 (n=79,403). The food-frequency questionnaire included report of brand names of supplements, enabling exact
calculations of the amounts of folic acid ingested per day. Dietary intake was adjusted for dietary folate equivalents (DFEs) when total intake was calculated.
c
Not quantified because reported daily energy intake was outside of the valid range, i.e., <4,500 kJ/day or >20,000 kJ/day.
d
Information on language skills was obtained from the MoBa 36-month questionnaire, and data were missing for autistic disorder cases whose mothers had not responded to that questionnaire (n=36).

JAMA. Author manuscript; available in PMC 2014 January 31.


Page 20